Method and apparatus for co-culturing cells

ABSTRACT

The method relates to the co-culturing of two cell cultures in two cell culture chambers such that the cellular products produced by the co-cultured cells are exchanged between the two cell culture chambers. The apparatus for culturing the cells contains two cell culture chambers separated by a microfiltration membrane which is permeable to medium, gases, and cellular products, and is impermeable to cells. A culture medium supply chamber is adjacent to and separated from the first cell culture chamber by a dialysis membrane which is permeable to medium and gases. The exterior portion of the second cell culture contains a gas-exchange membrane which allows gas exchange between the apparatus and the environment. The apparatus can be in a modular form where the first and second cell culture chambers form one module which is detachable from the culture medium supply chamber which forms the second module. The method and apparatus allows the exchange of media, gases, and cellular products between the two cell cultures while preventing direct contact of the two cell cultures.

BACKGROUND OF THE INVENTION

The invention relates to a method of cell culturing in which a cellproduct released from a first cell culture is transported into a secondcell culture and is integrated with the cells of the second cellculture.

This type of method may be used, for example, to transfer a cellproduct, such as a vector, from a first cell culture to a second cellculture, such as a lymphocyte culture. For this purpose, the first cellculture, for example a murine cell culture, which produces the vector ofinterest, will be cultured in a culture vessel. The cells will then beseparated by centrifugation from the released vectors and thesupernatant containing the vector will be added to the second cellculture, the lymnphocytes. In order to avoid thinning the lymphocyteculture too much, the addition of vectors is repeated several times. Itis also necessary to keep in mind that the half-life of the vectorslasts only 10 minutes, which, on the one hand, requires working asquickly as possible, and on the other hand, excludes the addition oflarge quantities of the vector in a single operation. For this reason,the known methods are slow.

In addition, because of the large amount of equipment required andnumerous steps involved in using the known methods, there is a constantrisk of contamination to the cell culture.

Moreover, the invention relates to a culture vessel for cell cultureshaving a first cell culture chamber for the reception of a first cellculture, having a supply chamber for the reception of a culture medium,having a dialysis membrane between the cell culture chamber and thesupply chamber by means of which the nutrients are transported into thecell culture chamber and the metabolic products of the cell culture areremoved from the cell culture chamber, and having a gas-exchangemembrane that forms at least part of the external portion of the culturevessel.

This type of culture vessel is known from DE-Al 42 29 325. It describesa culture vessel which has a supply chamber and at least one cellculture chamber within a substantially hollow, cylindrical plastichousing. The two chambers are separated from one another by a flat,semipermeable dialysis membrane. The culture medium is transported fromthe supply chamber into the cell culture chamber and the metabolicproducts removed from the cell culture chamber by means of the dialysismembrane. The end surface of the plastic tube facing the cell culturechamber is closed by a silicone foil approximately 0.5 mm in diameterthat serves as a gas-exchange membrane. The silicone foil is permeableto oxygen and carbon dioxide, but impermeable to liquids and bacteria.

The known culture vessel is able to rotate on a roller-rotator device onits long axis, by which means mixing of the liquid culture medium andcell culture plaque takes place in the cell culture chamber Oxygensupply to the cell culture is effected through the direct passage ofoxygen via the gas-exchange membrane to the cell culture plaque as wellas indirectly via prior passage to the nutrient solution, from which itreaches the cell culture chamber via the dialysis membrane.

The known culture vessel may have several cell culture chambers. It isconsequently capable of culturing one or more cell cultures. Thepreviously described method, referred to as co-culturing, however, isnot feasible by this means.

SUMMARY OF THE INVENTION

It is the object of this invention to offer a method for which the riskof contamination during transfer of a cell product to a second cellculture is reduced and which permits a high yield and rate ofproduction. Furthermore, it is the object of this invention to provide aculture vessel for cell cultures, in which a co-culturing method may beimplemented with a minimum of labor.

With regard to method, the object is solved in the context of theconvention by the previously mentioned method, in that the first andsecond cell cultures are contiguous, and in that they are separated by amembrane which is impermeable to the cells of the cell culture, butpermeable to the cell product, and in that the cell product istransported into the second cell culture via the membrane. Given thefact that the cell cultures are separated from one another only by themembrane, which is permeable to the cell product of the first cellculture, a continuous transfer of the cell product from one cell cultureto the other is made possible. Transfer of the cell product occurswithout a loss of time. The impetus for the transfer to the second cellculture chamber could, for example, be the equalization ofconcentration. A prolonged work period, such as for the separation ofthe cell product by centrifugation, and subsequent addition to thesecond cell culture is not required. The danger of accidental infectionof the cell cultures is thereby reduced. At the same rate at which thecell product is released from the first cell culture, it is drawnthrough the membrane and transported into the second cell culture.

The second cell culture can also be a cell-free medium. In this case,the cell product can be concentrated and removed without cellularbiomass. The second cell culture can also contain a second cell line,with which the cell product of the first cell line may be integrated.

Of proven value is a method in which the first and the second cellcultures are cultured in a shared culture medium, where they areseparated from the culture medium by a dialysis membrane that ispermeable to the nutrients. This method enables a continuous transfer ofthe cell product to the second cell culture over an extended period. Asneeded, the culture medium will be exchanged. The culture medium isseparable from the cell cultures by means of a semipermeable membranethat is permeable to the nutrients and the metabolic products of thecell cultures.

It has proven to be particularly advantageous to culture the first andthe second cell culture in a culture vessel that is sealed off from theoutside. The sealed culture vessel prevents contamination of the cellcultures. The culturing can be done in a roller bottle culture vessel.The rotating motion facilitates the mixing of the cell cultures andculture medium.

Of proven value is a method in which the first and/or the second cellculture is supplied with oxygen via a gas-exchange membrane. Thisfacilitates a contamination-free supply of oxygen to the cells. It isnot required that both cell cultures have direct contact with thegas-exchange membrane. The supply of oxygen can also occur indirectlythrough the passage of oxygen into one cell culture or into the culturemedium.

With regard to the device, the above-mentioned object is solved by theinvention previously described, in that there is a second cell culturechamber for the reception of a second cell culture, which is separatedfrom the first cell culture chamber by a filtration membrane that isimpermeable for the cells of the first and second cell culture.

Because the first cell culture chamber is separated from the second cellculture chamber by a filtration membrane, a transfer of the cellproducts from the first cell culture chamber is possible without fear ofcontaminating one or the other cell cultures. The transfer of cellproducts from one cell culture chamber to the second cell culturechamber can occur continuously. The invented culture vessel permitssimultaneous culturing of the two cell cultures side by side, forexample, in a common culture medium. As the filtration membrane isimpermeable to both cell cultures the contamination of the cell cultureswith cells of the other cell culture is excluded.

Of proven value is an embodiment of the culture vessel in which thefiltration membrane is a microfiltration membrane. Microfiltrationmembranes are available commercially. The diameter of the pores of themicrofiltration membrane can easily be synchronized with the size of thecell product. It is also of proven value to insert a dialysis membranewithin the filtration membrane.

It is an advantage to have a culture vessel in which the second cellculture chamber is bounded, on the one hand, by a filtration membrane,and on the other hand, by a gas-exchange membrane. In that the secondcell culture chamber is bounded by the gas-exchange membrane, the cellculture is especially well supplied with oxygen.

It is preferable for a culture vessel to have the dialysis membrane, thefiltration membrane, and the gas-exchange membrane constructedsubstantially on the same plane. Such membranes are easy to manufacture.The arrangement lends itself to a modularly-constructed culture vessel.In the case of at roller bottle culture vessel, the cell culturechambers could, for example, be arranged side by side along the longaxis of the culture vessel. However, the arrangement of one behind theother has proven very effective in being especially easy to produce.

Especially advantageous is an embodiment of the culture vessel which hasa supply module containing the supply chamber and a production modulecontaining the first and second cell culture chamber, and where themodules are detachable from one another. The supply chamber isconveniently provided with a one-way opening for pouring in the supplymedium. The modular construction of the culture vessel facilitatesreplacement of the individual modules. The cell culture chambers areconveniently provided with sealable openings. These may be used forfilling or removal of the cell cultures. These openings could, forexample, be embodied as septum or so-called Luer-Lock closures.

BRIEF DESCRIPTION OF THE DRAWINGS

One sample embodiment of the invention is shown in the figures and willbe further explained below. The figures show in detail

FIG. 1 a culture vessel, represented schematically, having aco-culturing module with two cell culture chambers and

FIG. 2 shows a cross-section of the portion of the co-culturing moduleindicated in FIG. 1 in the form of a technical drawing.

DETAILED DESCRIPTION OF THE INVENTION

The entire culture vessel is represented in FIG. 1 by the number 1. Theculture vessel 1 comprises three chambers, a supply chamber 2, a firstcell culture chamber 3 and a second cell culture chamber 4, The supplychamber 2 is, with the exception of an air bubble, filled with a culturemedium 5 for the cell cultures. Cell culture chamber 3 is divided fromthe supply chamber 2 by a flat dialysis membrane 6. Cell culture chamber4 is separated from cell chamber 3 by a microfiltration membrane 7. Cellculture chamber 4 is closed off from the outside by a gaspermeablesilicone membrane 10, with knobs which turn inward, are hollow and openoutwards for improved gas supply (see FIG. 2 below).

The cells in exemplary cell culture chamber 3 are murine fibroblasts 8.These release a cell product, namely a vector 9. The cells of the cellculture in exemplary cell culture chamber 4 are lymphocytes 11. Theseare able to incorporate into the cell nucleus the vector 9 released fromthe murine fibroblasts.

For filling and removing the cell cultures or the cell product, the endsurface of the roller bottle-type culture vessel 1, which has agas-exchange membrane 10, is provided with so-called Luer-Lock seals 13,and in the area of cell culture chamber 3, with a so-called septum 12 inthe cylinder housing surface of the culture vessel 1. For filling thesupply chamber 2 with the culture medium 5, there is a screw-cap 14located on the front side of the supply chamber 2. Otherwise, theculture vessel 1 is closed on all sides.

The culture vessel 1 comprises two modules, a supply module 15 with thesupply chamber 2, and a co-culturing module 16, which contains the twocell culture chambers, 3 ; 4. The two modules 15;16 are held together bya water-tight snap-lock 17 and are detachable.

To give a more detailed explanation of the method used in the invention.there follows a description of a method of gene therapy usingrecombinant lymphocytes with the aid of the culture vessel 1 shown inFIG. 1.

The lymphocytes 11 are human lymphocytes with a genetic defect, forexample, a disruption in the production of an enzyme required for thehuman organism. The murine fibroblasts 8 contain a retroviral vectorwith a gene which can replace the defective gene in the lymphocytes.They continuously release the vector 9, a virus or virus envelope. Asthe murine fibroblasts 8 grow tightly adherent, cell culture chamber 3is provided with an appropriate carrier, for example, microcarriers or aporous stationary matrix.

To implement the gene therapy, two cell lines are culturedsimultaneously or phased-in separately into cell chambers 3 and 4. Theinvented method will henceforth be referred to as the "co-culturingmethod." A phasing-in culturing method can, for example, be sensiblewhen in order to achieve an optimal "co-culturing" of both cell lines 8;11, one requires a head start on the other, or when the cell culturesneed different culture mediums for optimal growth.

In the "co-culturing" stage, both cell lines 8; 11 are suppliedsimultaneously with nutrients via the semipermeable membrane 6. For thispurpose, the pore diameter of the dialysis membrane 6 is set at aMWCO-value (Molecular Weight Cut-Off) of approximately 2 kilodaltons, sothat it is permeable both to the nutrients of the culture medium 5 andthe metabolic products of the cells 8 and 11, but riot to the murinefibroblasts 8 or the vector 9. In this way, the metabolic products canbe removed via the dialysis membrane 6 from the cell lines 8; 11 andsimultaneously the vector 9 can be concentrated in the co-culturingmodule 16. Via the gas-permeable membrane 10, the murine fibroblasts 8and the lymphocytes 11 are supplied with oxygen. The murine fibroblasts8, for which the cell density should not be too high, and thereforeshould be set at less than 10⁶ cells/ml, continuously release the vector9. An approximately 0.2 μm pore diameter should be selected for themicrofiltration membrane 7 so that the vector 9 can pass through themembrane, but not the cells of either cell line 8; 11. The result is acontinuous transfer of the vector 9 from cell culture chamber 3 intocell culture chamber 4. There, the intact gene, or a portion thereof, iscontinually flushed into the lymphocytes 11. This continuous method isespecially advantageous in the case where the vector 9 has only a shorthalf-life, perhaps only a few minutes.

Because of the short distances for the vector 9 and its rapid andcontinuous availability for the defective lymphocytes, there is a highrate of infection. The method runs in a closed system. Contamination ofthe medium 5 or the cell cultures 8; 11 is consequently avoided.

A further embodiment of the method of the invention is the case whencell culture chamber 4 is not filled with a cell culture. A dialysismembrane serves as a filtration membrane 7 between cell culture chamber3 which contains the murine fibroblasts 8, and cell chamber 4.Otherwise, the method and the culture vessel conform to the explanationand the embodiment explained above. By this method a cell-free harvestof the vector 9 from cell culture chamber 4 is possible.

In the following, an embodiment of a "co-culturing module" will beexplained in more detail using FIG. 2.

FIG. 2 shows a portion of a co-culturing module 16 enlarged by ratio of2:1. The co-culturing module 16 is formed from a circular span ring 18made of durable polycarbonate, on which are molded snap hooks 19, bymeans of which the co-culturing module 16 can be joined with the supplymodule 15. A silicone component 20 is inserted inside the span ring 18.The silicon component has areas of varying thickness. The thicker outerrim of the silicon component 20 is shown as number 35 in FIG. 2, andthat of the area covering the span ring is shown as number 18, which hasa thickness of approximately 0.5 mm, and that which serves as thegas-exchange membrane is shown as number 24. In order to enlarge thegas-exchange surfaces, the gas-exchange membrane 24 is provided withknobs opening outwards. Between the silicon component 20 and thedialysis membrane 6, a first cell culture chamber 23 is formed. Cellculture chamber 23 (the corresponding cell culture chamber is designatedas 4 in FIG. 1) holds a volume of approximately 35 ml and serves, forexample, for the reception and culturing of human lymphocytes containingdefective genes. The micro-filtration membrane 22 rests against thefirst lattice 25, whose rim 26 rests upon the silicone component 20. Thecross pieces of the lattice 25 serve, simultaneously, as a mixingelement during rotation of the co-culturing module 16.

A second cell culture chamber 28 is bounded by micro-filtration membrane22 on one side and a dialysis membrane 27 on the other side. It has avolume of approximately 5 ml and serves for the reception of murinefibroblasts (the corresponding cell culture chamber is designated as 3in FIG. 1) dialysis membrane 27 rests against a second lattice 29 whoserim 30 rests upon a middle silicone ring 31. The middle silicone ringprovides the thickness between the lattices, 25; 29 on the one hand, andthe septum for the cell culture chamber 28, on the other hand. Thesilicone component 20, the lattices 25; 29, the membranes 22; 27, andthe middle ring 31 are held together by the snap hooks 32, which areevenly distributed around the span ring 18.

For filling, emptying, inoculation or removal of samples from the cellculture chamber 23, Luer-lock closures 33 have been mounted on the spanring 18. The opening of the Luer-Lock closures 33 into cell chamber 23are sealed water-tight by means of caps.

We claim:
 1. A method of cell culturing comprising:(a) obtaining a cellculturing vessel comprising:(i) a first cell culture chamber and asecond cell culture chamber separated by a microfiltration membrane, forthe reception of a first and a second cell culture, respectively,wherein said microfiltration membrane allows exchange of culture medium,gases, selected cellular products, and metabolic waste products whilepreventing exchange of cells between said first and second cell culturechambers; (ii) a culture medium supply chamber adjacent to said firstcell culture chamber, said culture medium supply chamber and said firstcell culture chamber are separated by a dialysis membrane wherein saiddialysis membrane allows exchange of culture medium, gases, andmetabolic waste products, while preventing exchange of selected cellularproducts and cells between said first cell culture chamber and saidculture medium supply chamber; and (iii) a gas-exchange membrane thatforms at least a part of the exterior wall of said second cell culturechamber wherein said gas-exchange membrane allows the exchange of gasesbetween said second cell culture chamber and the surrounding atmosphere;(b) culturing a first cell culture in said first cell culture chamber;and (c) simultaneously culturing a second cell culture in said secondcell culture chamber.
 2. The method of cell culturing described in claim1 wherein the first and the second cell culture chambers are componentsof a roller bottle cell culture vessel.
 3. The method of cell culturingdescribed in claim 1 wherein the first and second cell cultures aresupplied with oxygen which passes through said gas-exchange membrane. 4.A cell culture vessel comprising:(a) a first cell culture chamber and asecond cell culture chamber separated by a microfiltration membrane, forthe reception of a first and a second cell culture, respectively,wherein said microfiltration membrane allows the exchange of culturemedium, gases, selected cellular products, and metabolic waste productswhile preventing exchange of cells between said first and second cellculture chambers; (b) a culture medium supply chamber for the receptionof a culture medium, said culture medium supply chamber is adjacent tosaid first cell culture chamber, and separated from said first cellculture chamber by a dialysis membrane, wherein said dialysis membraneallows exchange of culture medium, gases, and metabolic waste productswhile preventing exchange of selected cellular products and cellsbetween said first cell culture chamber and said culture medium supplychamber; and (c) a gas-exchange membrane which forms at least a part ofthe exterior wall of said second cell culture chamber wherein saidgas-exchange membrane allows the exchange of gases between said secondcell culture chamber and the surrounding atmosphere.
 5. The culturevessel as described in claim 4 wherein the second cell culture chamberis bounded by the microfiltration membrane and the gas-exchangemembrane.
 6. The culture vessel as described in claim 4 wherein thedialysis membrane, microfiltration membrane, and the gas-exchangemembrane are substantially aligned parallel to one another.
 7. Theculture vessel as described in claim 4 wherein the first and second cellculture chambers form a unitary structure which is capable of beingdetached from said culture medium supply chamber.
 8. The culture vesselas described in claim 4 wherein the first and second cell culturechambers have sealable openings.